This research proposal describes experiments designed to determine whether the protein Prp8 functions to coordinate the complex structural arrangement and/or rearrangement of RNAs at the spliceosomal catalytic core. To this end, the structural organization of Prp8 will be investigated by protease resistance mapping using epitope-tagged Prp8 in yeast extracts. As a complementary approach, a screen of transposon-mediated insertions that precisely divide Prp8 into two fragments will be used to identify independently functional, and complementary, domains of Prp8. The results of these experiments will be used to design portions of Prp8 for expression and purification, as well as full length Prp8, for biochemical analyses. Among these analyses is the exciting opportunity to determine whether Prp8 functions as an RNA chaperone to form or stabilize an RNA tertiary structure, in vitro that corresponds to an essential RNA interaction in vivo. These experiments will lay the foundation for further biochemical analyses of Prp8 and detailed studies of the mechanism of splicing catalysis.